Novel method for efficient purification of human serum albumin

ABSTRACT

The present invention describes a simple purification process for recombinant human serum albumin. The process results in highly purified protein with limited number of purification steps. The broth containing human albumin is clarified by centrifugation and microfiltration, diafiltered and captured by cation exchange chromatography by a process that allows 140-230 mg of albumin to be captured per mi of resin. Product related impurities are removed by hydrophobic interaction chromatography, optimised to allow 87-97% recovery in flow through mode. The final series of processes are so combined that there is easy transition from one step to the next with minimal interventions and adjustments. The entire process of purification is completed within two days from harvest to final product. Thus a cost-effective process with improved recovery of protein at each step is developed. The purified human serum albumin is analyzed for purity and shows physicochemical characteristics that are similar to standard albumin.

PRIORITY CLAIM

This application claims priority from the provisional applicationnumbered 3228/CHE/2014 filed with Indian Patent Office, Chennai on 1Jul. 2014 and postdated to 1 Jan. 2015 entitled “Novel method forefficient purification of human serum albumin”, the entirety of which isexpressly incorporated herein by reference.

PREAMBLE TO THE DESCRIPTION

The following specification particularly describes the invention and themanner in which it is to be performed:

DESCRIPTION OF THE INVENTION Technical field of the invention

The present invention discloses a simple, cost-effective purificationprocess of recombinant human serum albumin from different sources. Moreparticularly, the present invention relates to an improvement of themethod that enhances recovery of the protein is each step ofpurification.

Background of the invention

Human serum albumin is the most abundant soluble, globular, andunglycosylated monomeric protein in human plasma with a molecular weightof 66,437 to 66,600 Dalton. It contains a single unglycosylatedpolypeptide chain of 585 amino acids and also contains 17 disulphidebridges and a free thiol group. It acts as a carrier molecule and bindsto drugs, pigment, fatty acids, metal ions and to other proteins. Italso transports hormones, fatty acids, and other compounds, buffers pHand maintains osmotic pressure. Human serum albumin is used to replacelost fluid and help restore blood volume in trauma, burns and surgerypatients.

The market demand for human serum albumin is estimated as more than 500tons per year worldwide. Currently, commercial production of human serumalbumin is primarily based on collected human plasma, which is limitedin supply but of high clinical demand.

Recombinant human serum albumin is produced in yeasts including Pichia,Saccharomyces, Kluyveromyces, Hensenuela, rice and other organisms. Therecombinant protein could be produced by growing transgenic plants infields or greenhouses or by fermentation from different microorganisms.The protein thus produced needs to be purified through a series of stepsto finally attain a degree of purity that is equivalent to or betterthan plasma albumin. One of the biggest challenges in production ofalbumin is the degree of purity that is required to be attained. Whilemost recombinant proteins need to be purified such that the finalproduct has <100 ppm of host cell proteins (HCPs), recombinant albumin,which is used in high doses, is required to have <100 ppb (parts perbillion) of HCPs in the final product. Thus, a thousand-fold greaterpurity is required for recombinant albumin as compared to otherrecombinant bio therapeutics.

Purification of the protein to obtain, in pure form, and to homogeneity,free of: coloring material or pigment, host cell proteins, host cellDNA, polysaccharides, lipids, metal ions, degradation and aggregationproducts of albumin and glycated albumin. Additionally, the Cys 34 freethiol, which works as a free radical scavenger and is involved incarrying out anti-oxidant functions as well as in carrying drugs andother molecules needs to be maintained in its reduced form.

Since albumin tends to bind to many of the impurities present in thebroth the processes developed for purification of this protein arecomplicated, involving a large number of steps thus increasing the costwhile reducing the final recovery of the product.

There are many techniques described for purification of recombinanthuman serum albumin in patent and non-patent literature. However, thesemethods involve complicated and multiple steps, which may incuradditional cost for the process. The number of steps in prior artresults in reduced final recovery contributing to increase in cost ofthe product.

The Patent Application CN101768206 titled “Method for purifyingrecombinant human serum albumin and application thereof” describes amethod for purifying recombinant human serum albumin protein. Thismethod includes the steps of processing the fermented liquid containingrecombinant human serum albumin by a ceramic membrane, purifying thesupernatant liquid by high salt cation exchange chromatography,hydrophobic layer exchange chromatography and weak anion exchangechromatography. The protein obtained can be used for producing vaccinesfor humans against viruses with a cell culture method, particularlyrabies vaccines. However, the present invention is silent with respectto the purity and the recovery of the protein purified.

The Patent Application EP0570916 A2 titled “Recombinant human serumalbumin, process for producing the same and pharmaceutical preparationcontaining the same” describes purification of human serum albumin bysequence of steps including ultra-filtration, heat treatment, acidtreatment and another ultra-filtration, followed by subsequenttreatments with a cation exchanger, a hydrophobic chromatography carrierand an anion exchanger, and by salting-out such that a pure form ofhuman serum albumin is obtained which does not contain proteinaceous andpolysaccharide contaminants and is formulated into a pharmaceuticalpreparation. This process is efficient to purify recombinant human serumalbumin and to provide substantially pure human serum albumin, whichdoes not contain host-related substances and other contaminants and issufficiently free from coloration. However, the invention involves manysteps of purification with final recovery of the product being low.

The Patent Application EP0699687 B1 titled “Process for purifyingrecombinant human serum albumin” describes a process for purifyingrecombinant human serum albumin by heating a culture medium containingrecombinant human serum albumin and the host cells producing thisprotein, feeding the heated solution upwardly into a fluidized bed inwhich adsorbent particles are suspended to effect contacting with theadsorbent particles at a pH value of about 3 to 5 and then recoveringthe adsorbed fraction containing the recombinant protein. The solutionis heated in presence of a reducing agent and then subjected to at leastone purification treatment selected from a group consisting ofhydrophobic interaction chromatography, anion exchanger treatment,chelate resin treatment, boric acid/borate treatment andultra-filtration. The invention may result in increased purity of theprotein.

The Patent Application US20030204060 titled “Process for thepurification of serum albumin” describes purification of recombinanthuman serum albumin consisting of a series of steps, optionally byincubation with an anion-exchange adsorbent, followed by affinitychromatography employing a hydrophobic solid phase and using awater-soluble lipid anion as desorbents in the aqueous phase. Theimmobile phase comprises a carrier coupled to a 2-mercapto or 2-hydroxyalkanoic acid. The protein purified by this method is more than 99.9%pure, particularly more than 99.95% pure.

Thus, there is also a need for developing a process for purification ofrecombinant human albumin, which results in highly purified proteinthrough minimal steps while optimizing the process to increase theoutput from each step.

There is also a need for a purification process, which is cost-effectivefor large scale production of recombinant human serum albumin.

Objectives of the invention

It is the objective of the present invention to provide a process forpurification of recombinant human serum albumin in minimal number ofsteps.

It is another objective of the present invention to provide a processfor purification of recombinant human serum albumin which iscost-effective.

It is yet another objective of the present invention to provide aprocess for purification of recombinant human serum albumin whichresults in high purity of the recovered protein.

It is yet another objective of the present invention to optimize theprocess such that the protein is purified through minimal number stepswith increased output from each step.

It is yet another objective of the present invention to optimize theprocess through a sequence of steps that ensures completion ofpurification from clarification to bottling in less than two days.

SUMMARY OF THE INVENTION

The present invention discloses a simple, cost-effective purificationprocess for recombinant human serum albumin. The process results inhighly purified protein with limited number of purification steps. Thepresent invention also relates to an improvement of the method such thatrecovery of the protein is increased at each step of purification.

The recombinant human serum albumin produced by fermentation issubjected to purification. The fermentation broth is usually selectedfrom bacteria, fungi, mammalian cells or homogenate of transgenic plantproducing recombinant human albumin The cells are separated from thefermentation broth and subjected to centrifugation. The cell freesupernatant is microfiltered using 0.1-0.45 micron hollow fiber filters.The microfiltered sample is concentrated and the broth diafilteredagainst water using hollow fiber filters in a continuous mode till theconductivity is less than 3 mS/cm. The diafiltered sample is loaded on acation exchange column by online pH adjustment to 4.5 using 2% of 1MSodium acetate buffer. The eluent from this step is subjected tohydrophobic interaction chromatography, which employsPolypropyleneglycol (PPG), in a flow through mode.

The flow through and wash from purification on HIC is diafiltered andloaded on anion exchange resin for chromatography. The protein thuseluted is concentrated to 200 mg/mi and diafiltered against water. Thefinal concentrated product is brought to 20 mM phosphate buffer, pH 7.0,1.44 mM sodium chloride and 8 mM sodium caprylate by addition ofappropriate volumes of their stock solutions. The protein is sterilefiltered and bottled. The bottled protein is subjected to terminalpasteurization at 60° C. for 1-10 hrs.

The final series of process are combined so that there is simpletransition from one step to the next, thus reducing the overall time ofthe process. The entire process of purification is completed within twodays from harvest to final product.

The protein purified has a colorless to pale yellow color with a thiolratio of >0.75, aggregates <2%, physicochemical and bindingcharacteristics of the standard albumin.

The present invention makes the process commercially cost-effective.

BRIEF DESCRIPTION OF THE DRAWINGS

This invention is illustrated in the accompanying drawings.

FIG. 1 illustrates a flow chart of a process of purification ofrecombinant human serum albumin.

FIG. 2 illustrates the result of SDS PAGE of fractions obtained fromcation exchange chromatography loaded on 10% SDS PAGE gel and subjectedto coomassie staining.

FIG. 3 illustrates the separation of aggregates and degradation productson PPG as seen on SEC-HPLC using BioSep s2000 column.

FIG. 4 illustrates the different fractions obtained on fractionation ofhuman serum albumin on PPG loaded on 10% SDS-PAGE Gel. FIG. 4 also showsthe effect of adding caprylate and cysteine on enhanced recovery of theprotein in the flow through/wash.

FIG. 5 illustrates the SDS PAGE of the final purified product afterAnion exchange chromatography.

FIG. 6 illustrates the intact mass of the final recombinant human serumalbumin compared to standard albumin.

FIG. 7 illustrates Native PAGE, SDS-PAGE under reducing and non-reducingconditions and western blot of recombinant albumin hybridized toanti-human albumin antibody compared to standard albumin.

FIG. 8 illustrates the results of comparison of different types ofspectra of recombinant human serum albumin with standard albumin.

FIG. 9 illustrates the HPLC profiles of the final purified recombinanthuman serum albumin on SEC-HPLC and RP-HPLC.

FIG. 10 illustrates the manner of loading of broth after diafiltrationon cation exchange column.

FIG. 11 illustrates the manner of loading of broth, after heattreatment, on cation exchange column.

FIG. 12 illustrates the summary of process followed for cation exchangechromatography

FIG. 13 illustrates the consistent reduction of pigment at the end ofthe

Cation Exchange chromatography.

FIG. 14 illustrates the specifications of the final purified recombinanthuman albumin obtained using the described procedure for purification.

DETAILED DESCRIPTION OF THE INVENTION

In order to more clearly and concisely describe and point out thesubject matter of the claimed invention, the following definitions areprovided for specific terms, which are used in the following writtendescription.

The term “Recombinant human serum albumin” refers to human serum albuminproduced by recombinant DNA technology.

The term “Protein purification” refers to a series of processes intendedto isolate one or a few proteins from a complex mixture, usually cells,tissues or whole organisms or fermentation broth.

The term “SDS-PAGE” refers to a sodium dodecyl sulfate polyacrylamidegel electrophoresis (SDS-PAGE), a technique for separating proteinsbased on their ability to move within an electrical current, which is afunction of the length of their polypeptide chains or of their molecularweight.

The term “Cation Exchange Chromatography” refers to a form ofion-exchange chromatography that uses resins or packings with functionalgroups that separates cations. This may include filters with functionalgroups that separate cations

The term “Microfiltration” refers to a physical filtration process wherea fluid is passed through a membrane with pore size of 0.1-0.45 micronsto separate cells, cell debris, suspended particles or other componentswhich are larger than 0.1-0.45 microns in size, from process liquid.

The present invention discloses a process for purification ofrecombinant human serum albumin.

FIG. 1 illustrates a flow chart for a process of purification ofrecombinant human serum albumin. The process (100) of purificationstarts with (101) separation of cells from fermentation broth from anysource cultured using any organism. The fermentation broth is usuallyselected from the group consisting of bacteria, fungi, mammalian cellsor homogenate of transgenic plant producing recombinant human albumin.The broth is diluted with equal volume of water and centrifuged. Theprotein, can, optionally, be subjected to heat treatment at 60 C for 1-2hrs for viral inactivation or prevention of acid protease activitybefore proceeding to the next step of microfiltration. However, thisstep is optional.

At step (102), the cell free supernatant obtained after centrifugationis subjected to microfiltration using hollow fiber filters. Theresultant broth is completely free of particulate matter and celldebris. At step (103), the microfiltered sample is concentrated anddiafiltered against water to achieve the conductivity of less than 3mS/cm. At step (104), the diafiltered sample is purified by cationexchange chromatography. The pH of the sample is adjusted on-line to 4.5and loaded on cation exchange resin. The resident time of the protein isgradually increased so that a total of 140-230 mg of protein is loadedper ml of the resin. The protein is eluted with 60 mM sodium phosphate,pH 5.8 with 10 mM sodium caprylate. The addition of fatty acids such ascaprylate to the elution buffer significantly enhances recovery, reducesaggregation and results in pigment reduction. At step (105), the proteinis subjected to hydrophobic interaction chromatography, which employsPolypropylenglycol (PPG), a resin with different selectivity to theother HIC resins, to separate the aggregates and the 45 kDa degradationproduct from the recombinant human serum albumin. At step (106), theprotein pooled from the flow through and wash of the earlier step isconcentrated to 50 mg/ml and diafiltered against water. At step (107),the sample pH is adjusted to 7.0 and loaded on anion exchange resin forchromatography. At step (108), the protein is eluted from anion exchangeresin with sodium acetate, pH 4.5. The protein eluted is adjusted to pH7.0 and diafiltered against water. This protein is concentrated to 200mg/ml and diafiltered against water. Sodium phosphate buffer pH 7.0,sodium chloride and sodium caprylate are added to a final concentrationof 20 mM, 144 mM and 8 mM respectively. At step (109), the concentratedprotein is sterile filtered and bottled. The bottled protein issubjected to pasteurization at 60° C. for 1-10 hrs.

The final series of process are combined so that there is an easytransition from the last step to the next. This results in reduction oftime for completion of the process. The entire process of purificationis completed within two days from harvest to final product beingobtained. This makes the process cost effective and commercially viable.

FIG. 2 illustrates the result of Sodium Dodecyl Sulfate PolyacrylamideGel Electrophoresis (SDS PAGE) of fractions obtained from cationexchange chromatography loaded on 10% SDS PAGE gel and subjected tocoomassie staining. The diafiltered sample is loaded on a cationexchange column by online pH adjustment to 4.5 using 2% of 1M sodiumacetate buffer, pH 4.5. The protein is eluted with 60 mM Sodiumphosphate, pH 5.8 containing 10 mM Sodium caprylate. The fractions fromdifferent steps in the process including flow through, wash 1, wash 2,elution and regeneration are loaded on the gel.

FIG. 3 illustrates the separation of aggregates and degradation productson PPG, a HIC resin. 50 microliter of the flow through and regenerationsamples are injected into BipSep™ s2000 column for Size exclusionchromatography. The results showed the presence of monomeric human serumalbumin in the flow through while the regeneration fraction mainlycontains aggregates and degradation product.

FIG. 4 illustrates the different fractions obtained on fractionation ofhuman serum albumin on PPG loaded on 10% SDS-PAGE Gel. Left panelillustrates the results of chromatography without addition of caprylateand cysteine to the load while the panel on the right shows the resultsof addition of caprylate and

Cysteine to the load. Addition of caprylate and cysteine results inincreasing the amount of monomer in the flow through and wash from60-70% (without additives) to 87-97%. This process allows the separationof up to 30% aggregates and 30% degradation products in a single step.

FIG. 5 illustrates the SUS PAGE of the final purified product afterAnion Exchange chromatography. 20 micrograms of the protein have beenloaded on 10% SDS-PAGE and subjected to coomassie blue staining. Theresults show the purity of the recombinant human serum albumin.

FIG. 6 illustrates the intact mass of the final recombinant human serumalbumin. The intact mass of recombinant human serum albumin is 66.483kDa and is identical to that of standard HSA.

FIG. 7 illustrates Native PAGE, SUS-PAGE under reducing and non-reducingconditions and western blot and hybridization to anti-human albuminantibody of recombinant albumin compared to standard albumin. Theresults show identity between the recombinant human serum albuminpurified in the present invention to the standard albumin.

FIG. 8 illustrates the results of comparison of different types ofspectra of recombinant human serum albumin with standard albumin. Thepurified. recombinant human serum albumin is compared to standardalbumin by UV, fluorescence and CD spectroscopy. The results of thesespectra showed similarity of recombinant human serum albumin withstandard albumin.

FIG. 9 illustrates the HPLC profiles f standard and recombinant albumin.The purified recombinant human serum albumin is subjected to SEC HPLCand RP HPLC. The SEC HPLC showed high percentage of purity of proteinwith less that 1% of aggregates. The RP HPLC, showed 100% purity of therecombinant human serum.

The recombinant protein thus obtained in the above steps ischaracterized by mass spectrometry, which showed 100% purity. The Nterminal sequencing of the purified recombinant human serum albuminshowed identity to plasma albumin. The thiol ratio of the recombinantalbumin purified by the above process showed that more than 75% of themolecules have a free thiol group as compared to a maximum of 30% inplasma albumin. Since the free thiol is very important in the functionof albumin as a carrier molecule, the recombinant albumin provides asignificant advantage over the plasma albumin due to a higher percentageof the molecules comprising free thiol group.

The recombinant human serum albumin shows similar glycation,low-molecular weight impurities, binding characteristics to bilirubin,warfarin and fatty acid as standard albumin.

In order that this invention to be more fully understood the followingpreparative and testing examples are set forth. These examples are forthe purpose of illustration only and are not to be construed as limitingthe scope of the invention in any way.

EXAMPLE 1 Clarification

-   -   a. The first step of the purification of the protein is cell        separation. The fermentation broth is diluted with equal volume        of water and subjected to centrifugation at 5000 rpm for 5        minutes. This step results in recovery of >90% of the protein.    -   b. The cell free supernatant isolated in the previous step is        subjected to microfiltration with filters of 0.1-0.45 microns        pore size. >90% of protein is recovered with the filtrate being        completely free of cells, cell debris and particulate matter.

EXAMPLE 2 Diafiltration

The microfiltered broth from Example 1 is subjected to diafiltration.The filtrate is concentrated to 20 mg/ml and diafiltered against waterusing 30 kDa hollow fiber filters till the conductivity is <2 mS/cm. Thediafiltered sample is used for column chromatography. The recovery inthis step is >90%.

An optional step of heat treatment can be introduced aftermicrofiltration, wherein 5-20 mM sodium caprylate and 5-20 mM cysteineare added to the broth and the samples are heated to 60° C. for 60-120minutes. This step is effective in denaturation of acid proteases, ifany, that would otherwise cause degradation of the protein. However,this step is only optional and not essential to achieve the purificationof recombinant albumin.

EXAMPLE 3 Cation Exchange Chromatography

SP Sepharose FF, a cation exchange resin that is both cheap and haslongevity—the resin has been tested for 300 cycles, is packed to a bedheight of 15 cm. It is equilibrated with 50 mM sodium acetate buffer, pH4.5. The diafiltered sample at neutral pH, is diluted with water to 10mg/ml. The protein is loaded onto the column with on-line pH adjustmentto 4.5 using 2% 1M sodium acetate, pH 4.5. The resident time of theprotein is gradually increased to obtain maximum binding of albumin tothe resin. The loading is performed as shown in FIG. 10.

Under the above conditions, 230 mg of albumin is loaded per ml of theresin. The flow through or equilibration wash does not have any tracesof albumin. SP Sepharose FF from GE is claimed to exhibit a bindingcapacity of 130 mg/ml for BSA at 10% breakthrough, this method ofbinding enables 75% more binding to the resin thus reducing the cost pergram of the final product.

Further, the samples which are heat treated after diafiltration arediluted to 5 mg/ml with water and loaded onto the column with on-line pHadjustment to 4.5 as shown in FIG. 11.

Under the above conditions, up to 140 mg of albumin is loaded per ml ofthe resin without any breakthrough. Binding has also been done whereinalbumin has been bound up to 80 mg/ml of resin at 5 minutes residenttime followed by loading up to 140 mg/ml at 15 minutes resident time.140 mg albumin/ml of resin is successfully bound under these conditionswith no traces of albumin in the flow through

The process followed after loading is summarized in FIG. 12.

After loading, the column is washed with equilibration buffer, followedby washing with 3CVs of 25 mM sodium acetate, pH 4.5 containing 50 mMammonium sulfate and 2% Tween 20. This step of washing removes pigment,host cell protein and certain degradation products of albumin. This isfollowed by washing with 2-3 CVs of 25 mM sodium acetate, pH 4.5followed by 3 CVs of 25 mM sodium acetate, pH 4.5 containing 2M Urea.This step of washing removes pigment very effectively. Urea is removedfrom the column by washing with 3CVs of 25 mM sodium acetate, pH 4.5.

The protein is eluted with 60mM sodium phosphate buffer, pH 5.8 with 10mM sodium caprylate. The protein recovered in this step is >90%.

Other cation exchange resins may also be used in the place of SPSepharose FF used in this example. Cation exchangers with claims ofhigher binding capacities will, using the above method of loading, beable to give 75% to two fold higher binding capacity for albumin ascompared to the claim by the manufacturer.

The addition of caprylate enhances recovery of the protein and reducesaggregation. Consistent reduction of pigment is seen at the end of theCation Exchange chromatography as tabulated in FIG. 13.

EXAMPLE 4 Hydrophobic Interaction Chromatography

The protein purified by Example 3 comprises albumin, its aggregates andits 45 kDa degradation product. The protein is subjected to hydrophobicinteraction chromatography in the flow through mode using PPG as theresin of choice.

To the eluent from the previous step, ammonium sulfate is added to afinal concentration of 1.2M. Cysteine is added to a final concentrationof 10 mM. The pH is adjusted to 7.0 and caprylate concentrationreadjusted to 10 mM with further addition of desired volume of 1M sodiumcaprylate. The prepared sample is loaded on the HIC column.

PPG resin is packed to a bed height of 15 cm and equilibrated with 25 mMPhosphate buffer, pH 7.0 with 1.2M ammonium sulfate, 10 mM Cysteine and10 mM Caprylate. The sample prepared above is loaded on the column at aresident time of 15 minutes. Monomeric albumin does not bind under theabove conditions while the 45 kDa degradation product and aggregates ofalbumin bind to the column. The resin has a binding capacity of 15mg/ml. Hence up to 150 mg albumin/ml resin is loaded on the column whenthe sum of aggregates and degradation products of albumin are <10% ofthe total albumin. If the percentage of aggregates and degradationproducts of albumin exceeds 10%, the amount of albumin to be loaded isadjusted accordingly.

Most of the monomeric albumin flows out of the column. A wash with 3-5CVs of equilibration buffer removes the rest of the monomeric albumin,which is loosely bound to the resin. The aggregates and degradationproducts are eluted with water.

The flow through and wash is pooled together. The recovery in this stepis 87-97%. The addition of caprylate results in >87% recovery of monomerwhile in its absence recovery achieved is <70%. Cysteine helps inkeeping the single thiol “free” and reduces the pigment in the finalproduct.

The aggregates at the end of this step are <2.5%, free thiol >70% andA350/A280=0.03-0.035. No traces of degradation products are seen in thefinal pool.

EXAMPLE 5 Diafiltration

The flow through and the wash obtained is concentrated to >50 mg/ml anddiafiltered against water till conductivity is <2-3 mS/cm. The recoveryin this step is 95-98%.

EXAMPLE 7 Anion Exchange Chromatography

The anion exchange resin (DEAE Sepharose Fast Flow or any other anionexchange resin) is packed to a bed height of 15 cm and equilibrated with20 mM Phosphate buffer, pH 7.0. The protein obtained in Example 5 isdiluted to 5 mg/ml with water and loaded on anion exchange resin. Thecolumn is washed with 5 CVs of equilibration buffer followed by elutionwith 50 mM sodium acetate pH 4.5. This step results in recovery of98-100% of protein.

The aggregates at the end of this step are <2.5%, free thiol 0.75-0.80.The pigment reduces to A350/A280=0.025−0.015.

EXAMPLE 8 Concentrating the Protein

The protein eluted from Example 7 is adjusted to pH 7.0 with sodiumhydroxide. The protein is concentrated to 200 mg/ml and diafilteredagainst water. Phosphate buffer, pH 7.0, sodium chloride and sodiumcaprylate are added to a final concentration of 20 mM sodium phosphatebuffer, pH 7.0. 144 mM sodium chloride and 8mM sodium caprylaterespectively. The protein is filtered through 0.2 micron filters intothe storage bottles. The bottled protein is subjected to pasteurizationat 60° C. for 10 hrs. The final product has the characteristics as shownin FIG. 14.

The physicochemical characteristics of the albumin are identical to thestandard albumin as determined by Mass spectrophotometry, N terminalsequencing, C terminal sequencing, IEF, 2 D IEF, UV, Fluorescence and CDspectra and binding characteristic for bilirubin, warfarin and fattyacids.

The protein purified by the process of the present invention results inhigh purity of recombinant human serum albumin. The entire process ofpurification is completed within two days from harvest to final productbeing obtained. This makes the process commercially successful andviable. Hence the process is cost-effective.

We claim:
 1. A process for purification of recombinant human albumin,the process (100) comprising the steps of: a. separating plurality ofcells from fermentation broth or harvest by centrifugation (101); b.microfiltering the obtained cell free supernatant (102); c.concentrating the microfiltered and diafiltering the broth against water(103); d. loading the diafiltered sample on a cation exchange columnpurification in bind and elute mode (104); e. separating the monomericalbumin from aggregates and degradation products by hydrophobicinteraction chromatography in a flow through mode (105); f. pooling theflow-through and wash and diafiltering the same against water (106); g.loading the albumin on anion exchange resin for chromatography in bindand elute mode (107); h. concentrating the eluted protein anddiafiltering against water (108); and i. sterile filtering the proteinand subjecting to pasteurization at 60° C. for 1-10 hours (109).
 2. Theprocess as claimed in claim 1, wherein the purity of recombinant humanalbumin is greater than 97% with reduced pigment and free thiol ratio ofgreater than 0.75, one or more aggregates less than 2.5% and degradationless than 2% within 60 hrs from harvesting to bottling of recombinanthuman albumin.
 3. The process as claimed in claim 1, whereinfermentation broth is selected from the group consisting of bacteria,fungi, mammalian cells or homogenate of transgenic plant producingrecombinant human albumin.
 4. The process as claimed in claim 1, whereinloading cation exchange resin by gradual increase in resident timeenables increasing the loading capacity by 75-150% above the labelledbinding capacity of the resin.
 5. The process as claimed in claim 1,wherein addition of caprylate and cysteine during hydrophobicinteraction chromatography results in increasing the recovery of theprotein to >87%.
 6. The process as claimed in claim 1, whereinHydrophobic Interaction Chromatography (HIC) in flow through modeemploys addition of cysteine and one or more fatty acids.
 7. The processas claimed in claim 1, wherein caprylate at the concentration of 5-30 mMenhanced recovery of the monomeric albumin and the recovery of albuminin the pool of the flow through and wash is ≧87% in HIC in flow throughmode.
 8. The process as claimed in claim 1, wherein 5-30 m M cysteine isadded to achieve free thiol ratio of albumin at greater than 0.7 in HICin flow through mode.
 9. The process as claimed in claim 1, whererecombinant human albumin exhibited improved purity, thiol ratio andpigmentation.
 10. The process as claimed in claim 1, wherein smoothloading of protein samples results in reduction in time thus making theprocess cost-effective.